High performance liquid chromatography (HPLC) is a very popular separation technique in analytical sciences. Additional information about HPLC can be found e.g., in “HPLC:Practical and Industrial Applications” by Joel K. Swadesh, 2nd ed., CRC Press Boca Raton, Fla. 2000; “Advances in Chromatography” by Eli Grushka and Nely Grinberg, 1st ed., CRC Press, Boca Raton, Fla., 2007); each of these references, is incorporated herein by reference.
Manufacturing HPLC instruments and accessories is a two billion dollars plus business annually and impacts many industries, including pharmaceutical, chemical, food, and environmental markets. Traditionally, based on the properties of the analytes to be separated from a sample, different types of HPLC can be applied; for example, reversed-phase liquid chromatography for analytes with hydrophobic groups, especially neutral or non-ionized compounds; and normal-phase liquid chromatography for polar compounds.
A limitation of current technology is that for unknown mixtures or complex samples containing compounds of different chemical properties in terms of hydrophobicity, polarity, and ionizability, one single type of HPLC may not be sufficient to resolve all the compounds. There are currently two possible solutions in the market for the separation of complex mixtures, such as separation of various metabolites from a mixture, e.g., to be able to analyze the metabolites individually and understand the metabolism.
One approach is two or multi-dimensional separation, in which more than one separation mechanism is employed. This method requires e.g., two or more columns to be utilized together or coupled, each separating a different compound or analyte from a sample. This method results in more instrumentation, more time in analysis, and considerably more expense.
Another approach lies in the improvement of the liquid chromatography (LC) columns. Scientists have tried to embed polar groups in the stationary phase of reversed-phased columns and by this method combine two separation mechanisms within one column. These are commercially available and have become quite popular since their introduction. However, as these existing methods rely on special chemical modifications they lack flexibility, and their efficiency is still under debate.
Effective separation of complex mixtures is a problem, the solution for which could have considerable commercial impact.